Interpretive Summary: Sorghum serves as an important forage crop for US as well as a model for bioenergy crop development. Brown midrib 6 (bmr-6), which has been commonly used in breeding for improved forage quality, is a mutant that affects lignin production (a cell wall polymer). In this study, we identified the mutation causing bmr-6 and showed that the mutation affects an enzyme involved in lignin synthesis. We have characterized the function of this enzyme and determined that this enzyme is completely non-functional in bmr-6 plants. bmr-6 represents a strategic target to improve forage quality for bioenergy uses.

Technical Abstract:
Brown midrib 6 (bmr-6) affects phenylpropanoid metabolism resulting in reduced lignin concentrations and altered lignin composition in Sorghum bicolor. Recently, bmr-6 plants were shown to have very limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of phenolic aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr-6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr-6 protein was absent in protein extracts from bmr-6 plants. Phylogenetic analysis indicated that Bmr-6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr-6 is distinct from the other CAD-like proteins in sorghum including SbCAD4 (Sb02g024190). Although both Bmr-6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr-6 and SbCAD4 showed that Bmr-6 had 1-2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr-6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His57, which is unique to Bmr-6 and other grass CADs. In summary, Bmr-6 encodes the major CAD protein involved in lignin synthesis in Sorghum bicolor, and the bmr-6 mutant is a null allele.